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See detailUV Spectroscopy of DNA Duplex and Quadruplex Structures in the Gas Phase
Rosu, Frédéric ULg; Gabelica, Valérie ULg; De Pauw, Edwin ULg et al

in Journal of Physical Chemistry A (2012), 116

UV absorption spectroscopy is one of the most widely used methods to monitor nucleic acid folding in solution, but the absorption readout is the weighted average contribution of all species present in ... [more ▼]

UV absorption spectroscopy is one of the most widely used methods to monitor nucleic acid folding in solution, but the absorption readout is the weighted average contribution of all species present in solution. Mass spectrometry, on the other hand, is able to separate constituents of the solution based on their mass, but methods to probe the structure of each constituent are needed. Here, we explored whether gas-phase UV spectroscopy can give an indication of DNA folding in ions isolated by electrospray mass spectrometry. Model DNA single strands, duplexes, and G-quadruplexes were extracted from solution by electrospray; the anions were stored in a quadrupole ion trap and irradiated by a tunable laser to obtain the UV action spectra of each complex. We found that the duplex and quadruplex spectra are significantly different from the spectra of single strands, thereby suggesting that electronic spectroscopy can be used to probe the DNA gas-phase structure and obtain information about the intrinsic properties of high-order DNA structure. [less ▲]

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See detaild(TG(n)T) DNA sequences do not necessarily form tetramolecular G-quadruplexes.
Joly, Laure; Rosu, Frédéric ULg; Gabelica, Valérie ULg

in Chemical Communications (2012), 48(67), 8386-8

Contrasting with the common belief that d(TG(n)T) DNA sequences would form tetramolecular G-quadruplex assemblies, mass spectrometry reveals that these sequences tend to fold into G-quadruplex trimers ... [more ▼]

Contrasting with the common belief that d(TG(n)T) DNA sequences would form tetramolecular G-quadruplex assemblies, mass spectrometry reveals that these sequences tend to fold into G-quadruplex trimers, dimers, and eventually monomers as the G-tract length increases. The final structure also depends on the ionic strength. [less ▲]

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See detailDisulfide bond assignement and folding characterization of peptide toxins by Ion Mobility Mass Spectrometry
Echterbille, Julien ULg; Quinton, Loïc ULg; Rosu, Frédéric ULg et al

Conference (2011, October 11)

Main component of animal venoms is peptide toxins, which are highly structured by several disulfide bridges. Disulfide bridges fill different roles as increasing the toxins efficiency by lowering their ... [more ▼]

Main component of animal venoms is peptide toxins, which are highly structured by several disulfide bridges. Disulfide bridges fill different roles as increasing the toxins efficiency by lowering their immunogenicity or providing the adequate conformation to efficiently bind to the biological receptor. The sequencing and the determination of the cysteine pairing is still challenging and therefore an important step in structural analysis. In this work, we present a new strategy to sequence structured toxins and assign S-S bridges using ion mobility resolved MS/MS. The method relies on the analysis of partially reduced multiple-disulfide peptide. The mixture of the different forms is resolved by ion mobility, followed by MS/MS acquisition on each mobility separated species. The proof of concept has been successfully conducted on α-CnI, a toxin purified from the venom of Conus consors marine snail. The toxin’s sequence contains four cysteines linked together with two disulfide bridges. α-CnI was partially reduced by a small excess of tris(carboxyethyl)phosphine (10:1). The resulting mixture was purified before analysis by infusion nanoESI-Synapt-G2. Fragmentation was performed after the mobility cell, to obtain specific fragments of each species. Partial reduction of α-CnI results in a mixture of oxidized (the two disulfides are formed), reduced (the two disulfides have been reduced) and partially reduced forms (one of the two disulfides has been reduced). The arrival time distribution of triply charged ions reveals the presence of 4 different species, characterized by different relative cross sections in the gas-phase. Mass matching allows identifying the species: the first mobility (the most compact structure) was identified to be the oxidized folded toxin (M). The latest peak, corresponding to the larger cross-section, was identified as the fully reduced toxin (M+4Da). The second and the third mobility peaks were attributed to the two partially reduced forms in which only one disulfide bridge was reduced (M+2Da). The change in ion mobility depends on which S-S bridge is reduced. Ion mobility separated species give characteristic fragment ions upon fragmentation in the transfer cell (i.e. after ion mobility separator). Interestingly, fragment ions coming from partially reduced species, especially the C-S or S-S bond cleavages, clearly indicates that the disulfide linkage of α-CnI is (Cys1-Cys3) and (Cys2-Cys4) as expected from literature. The method is now being applied with success to more complex systems containing 3 or 4 disulfide bridges. The influence of the charge state on the mobility separation is systematically analyzed in terms of structural implications. [less ▲]

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See detailDisulfide bonds assignment and folding characterization of peptide toxins by Ion Mobility Mass Spectrometry
Echterbille, Julien ULg; Quinton, Loïc ULg; De Pauw, Edwin ULg et al

Conference (2011, April 29)

Main component of animal venoms is peptide toxins, which are highly structured by several disulfide bridges. Disulfide bridges fill different roles as increasing the toxins efficiency by lowering their ... [more ▼]

Main component of animal venoms is peptide toxins, which are highly structured by several disulfide bridges. Disulfide bridges fill different roles as increasing the toxins efficiency by lowering their immunogenicity or providing the adequate conformation to efficiently bind to the biological receptor. The sequencing and the determination of the cysteine pairing is still challenging and therefore an important step in structural analysis. In this work, we present a new strategy to sequence structured toxins and assign S-S bridges using ion mobility resolved MS/MS. The method relies on the analysis of partially reduced multiple-disulfide peptide. The mixture of the different forms is resolved by ion mobility, followed by MS/MS acquisition on each mobility separated species. The proof of concept has been successfully conducted on α-CnI, a toxin purified from the venom of Conus consors marine snail. The toxin’s sequence contains four cysteines linked together with two disulfide bridges. α-CnI was partially reduced by a small excess of tris(carboxyethyl)phosphine (10:1). The resulting mixture was purified before analysis by infusion nanoESI-Synapt-G2. Fragmentation was performed after the mobility cell, to obtain specific fragments of each species. Partial reduction of α-CnI results in a mixture of oxidized (the two disulfides are formed), reduced (the two disulfides have been reduced) and partially reduced forms (one of the two disulfides has been reduced). The arrival time distribution of triply charged ions reveals the presence of 4 different species, characterized by different relative cross sections in the gas-phase. Mass matching allows identifying the species: the first mobility (the most compact structure) was identified to be the oxidized folded toxin (M). The latest peak, corresponding to the larger cross-section, was identified as the fully reduced toxin (M+4Da). The second and the third mobility peaks were attributed to the two partially reduced forms in which only one disulfide bridge was reduced (M+2Da). The change in ion mobility depends on which S-S bridge is reduced. Ion mobility separated species give characteristic fragment ions upon fragmentation in the transfer cell (i.e. after ion mobility separator). Interestingly, fragment ions coming from partially reduced species, especially the C-S or S-S bond cleavages, clearly indicates that the disulfide linkage of α-CnI is (Cys1-Cys3) and (Cys2-Cys4) as expected from literature. The method is now being applied with success to more complex systems containing 3 or 4 disulfide bridges. The influence of the charge state on the mobility separation is systematically analyzed in terms of structural implications. [less ▲]

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See detailDisulfide bond assignment and folding characterization of peptide toxins by Ion Mobility Mass Spectrometry
Echterbille, Julien ULg; Quinton, Loïc ULg; Rosu, Frédéric ULg et al

Poster (2011)

Introduction Animal venoms are mainly composed of peptide toxins, which are highly structured by several disulfide bridges. Disulfide bridges are a key feature as (i) they increase the toxins efficiency ... [more ▼]

Introduction Animal venoms are mainly composed of peptide toxins, which are highly structured by several disulfide bridges. Disulfide bridges are a key feature as (i) they increase the toxins efficiency by lowering their immunogenicity; (ii) they provide the adequate conformation for high affinity binding to the biological receptor. The sequencing and the determination of the cysteine pairing is still challenging and therefore an important step in their structure analysis and the understanding of their interactions with receptors. In this work, we present a new strategy to sequence structured toxins and assign S-S bridges using ion mobility resolved MS/MS. Methods The method relies on the analysis of partially reduced multiple-disulfide peptide. The mixture of the different forms is resolved by ion mobility, followed by MS/MS acquisition on each mobility separated species. The proof of concept has been successfully conducted on α-CnI, a toxin purified from the venom of Conus consors marine snail. The toxin sequence is GRCCHPACGKYYSC-NH2. It contains four cysteines linked together with two disulfide bridges. α-CnI was partially reduced by a small excess of tris(carboxyethyl)phosphine (10:1) at 56°C during 30min. The resulting mixture was purified by ZipTip C18 micro columns before analysis by infusion nanoESI-Synapt-G2. Fragmentation was performed after the mobility cell, to obtain specific fragments of each species. Mobilograms and mass spectra were analyzed using MassLynx (v4.1) and Driftscope (v2.1) from Waters. Preliminary data Partial reduction of a-CnI was performed in order to obtain a mixture of oxidized (the two disulfides are formed), reduced (the two disulfides have been reduced) and partially reduced forms (only one of the two disulfides has been reduced). The arrival time distribution of triply charged ions reveals the presence of 4 different species, characterized by a different relative cross sections in the gas-phase. The charge state of the ions influences the ion mobility separation. Mass matching allows identifying the species: the first mobility (the most compact structure) was identified to be the oxidized folded toxin (M=1541.58 Da). The latest peak, corresponding to the larger cross-section, was identified as the fully reduced toxin (M=1545.6 Da). The second and the third mobility peaks were attributed to the two partially reduced forms in which only one disulfide bridge was reduced (M=1543.59 Da). The change in ion mobility depends on which S-S bridge is reduced. Ion mobility separated species give characteristic fragment ions upon fragmentation in the transfer cell (i.e. after ion mobility separator). Interestingly, fragment ions coming from partially reduced species, especially the C-S or S-S bond cleavages, clearly indicates that the disulfide linkage of α-CnI is (Cys1-Cys3) and (Cys2-Cys4) as expected from literature. The method is now being applied with success to more complex systems containing 3 or 4 disulfide bridges. The influence of the charge state on the mobility separation is systematically analyzed in terms of structural implications. Novel aspect Sequencing and disulfide bridges assignment of peptide toxins using ion mobility resolved MS/MS [less ▲]

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See detailTridentate N-Donor Palladium(II) Complexes as Efficient Coordinating Quadruplex DNA Binders
Largy, Eric; Hamon, Florian; Rosu, Frédéric ULg et al

in Chemistry : A European Journal (2011), 17(47), 13274-13283

Fifteen complexes of palladium, platinum, and copper, featuring five different N-donor tridentate (terpyridine-like) ligands, were prepared with the aim of testing their G-quadruplexDNA binding properties ... [more ▼]

Fifteen complexes of palladium, platinum, and copper, featuring five different N-donor tridentate (terpyridine-like) ligands, were prepared with the aim of testing their G-quadruplexDNA binding properties. The fluorescence resonance energy transfer melting assay indicated a striking positive effect of palladium on G-quadruplex DNA stabilization compared with platinum and copper, as well as an influence of the structure of the organic ligand. Putative binding modes (noncoordinative p stacking and base coordination) of palladium and platinum complexes were investigated by ESI-MS and UV/Vis spectroscopy experiments, which all revealed a greater ability of palladium complexes to coordinate DNA bases. In contrast, platinum compounds tend to predominantly bind to quadruplex DNA in their aqua form by noncoordinative interactions. Remarkably, complexes of [Pd(ttpy)] and [Pd(tMebip)] (ttpy=tolylterpyridine, tMebip=2,2'-(4-p-tolylpyridine-2,6-diyl)bis(1-methyl-1H-benzo[d]imidazole)) coordinate efficiently G-quadruplex structures at room temperature in less than 1 h, and are more efficient than their platinum counterparts for inhibiting the growth of cancer cells. Altogether, these results demonstrate that both the affinity for G-quadruplex DNA and the binding mode of metal complexes can be modulated by modifying either the metal or the organic ligand. [less ▲]

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See detailTetramolecular G-quadruplex formation pathways studied by electrospray mass spectrometry
Rosu, Frédéric ULg; Gabelica, Valérie ULg; Poncelet, Harmonie et al

in Nucleic Acids Research (2010)

Electrospray mass spectrometry was used to investigate the mechanism of tetramolecular G-quadruplex formation by the DNA oligonucleotide dTG5T, in ammonium acetate. The intermediates and products were ... [more ▼]

Electrospray mass spectrometry was used to investigate the mechanism of tetramolecular G-quadruplex formation by the DNA oligonucleotide dTG5T, in ammonium acetate. The intermediates and products were separated according to their mass (number of strands and inner cations) and quantified. The study of the temporal evolution of each species allows us to propose the following formation mechanism. (i) Monomers, dimers and trimers are present at equilibrium already in the absence of ammonium acetate. (ii) The addition of cations promotes the formation of tetramers and pentamers that incorporate ammonium ions and therefore presumably have stacked guanine quartets in their structure. (iii) The pentamers eventually disappear and tetramers become predominant. However, these tetramers do not have their four strands perfectly aligned to give five G-quartets: the structures contain one ammonium ion too few, and ion mobility spectrometry shows that their conformation is more extended. (iv) At 4°C, the rearrangement of the kinetically trapped tetramers with presumably slipped strand(s) into the perfect G-quadruplex structure is extremely slow (not complete after 4 months). We also show that the addition of methanol to the monomer solution significantly accelerates the cation-induced G-quadruplex assembly. [less ▲]

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See detailElectronic spectroscopy of nucleic acids in the gas phase
Gabelica, Valérie ULg; Rosu, Frédéric ULg; Joly, Laure ULg et al

Conference (2010)

DNA polyanions trapped in a mass spectrometer undergo electron detachment following UV irradiation. Photodetachment is a single-photon process. Its efficiency depends on the nature of the DNA bases, the ... [more ▼]

DNA polyanions trapped in a mass spectrometer undergo electron detachment following UV irradiation. Photodetachment is a single-photon process. Its efficiency depends on the nature of the DNA bases, the ion's charge, and the excitation wavelength. Photodetachment can therefore be used to perform ion spectroscopy experiments, which probe electronic excitation within the initial charge state of the nucleic acids. Ion spectroscopy experiments on trapped nucleic acid cations and anions were performed from 4 to 20 eV using an OPO laser or using synchrotron radiation. Photoelectron spectroscopy experiments were also performed on multiply charged anions to probe direct detachment cross sections and electronic excitations within the final charge. The electronic spectra obtained from photodetachment integral cross sections show several resonances, provided that the photon energy is larger than the electron binding energy. We will also discuss whether the electronic spectra obtained via photodetachment can be used to probe gas phase ion structure. [less ▲]

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See detailInternal energy content of ions in a travelling wave ion guide
Morsa, Denis ULg; Gabelica, Valérie ULg; Rosu, Frédéric ULg et al

Conference (2010)

Travelling wave ion guides (TWIGs) separate of ions according to their mobility and, at constant charge, according to their shape. The ion mobility separation itself should not modify the shape of the ... [more ▼]

Travelling wave ion guides (TWIGs) separate of ions according to their mobility and, at constant charge, according to their shape. The ion mobility separation itself should not modify the shape of the systems investigated. It was recently suggested by Shvartsburg et al. that the higher fields used in TWIGs than in traditional drift tubes would cause significant heating of the ions. We present a quantitative analysis of the amount of internal energy imparted to ions as they are separated in TWIGs. Benzylpyridinium ions were chosen as “thermometer” ions. Based on arrival time distributions, the fragment ion population is separated as a function of the place of formation: before, in, or after the TWIG. Fragmentation all along the TWIG was observed. The roles of the travelling wave’s voltage, speed, and of the gas nature and pressure will be discussed in detail as well of the consequences on instruments performances. [less ▲]

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See detailCation Involvement in Telomestatin Binding to G-Quadruplex DNA
Rosu, Frédéric ULg; Gabelica, Valérie ULg; Smargiasso, Nicolas ULg et al

in Journal of Nucleic Acids (2010)

The binding mode of telomestatin to G-quadruplex DNA has been investigated using electrospray mass spectrometry, by detecting the intact complexes formed in ammonium acetate. The mass measurements show ... [more ▼]

The binding mode of telomestatin to G-quadruplex DNA has been investigated using electrospray mass spectrometry, by detecting the intact complexes formed in ammonium acetate. The mass measurements show the incorporation of one extra ammonium ion in the telomestatin complexes. Experiments on telomestatin alone also show that the telomestatin alone is able to coordinate cations in a similar way as a crown ether. Finally, density functional theory calculations suggest that in the G-quadruplex-telomestatin complex, potassium or ammonium cations are located between the telomestatin and a G-quartet. This study underlines that monovalent cation coordination capabilities should be integrated in the rational design of G-quadruplex binding ligands. [less ▲]

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See detailElectrospray Mass Spectrometry of Telomeric RNA (TERRA) Reveals the Formation of Stable Multimeric G-Quadruplex Structures
Collie, Gavin W.; Parkinson, Gary N.; Neidle, Stephen et al

in Journal of the American Chemical Society (2010), 132(27), 93289334

We report on the self-assembled structures formed by 12-mer, 22-mer, and 45-mer telomeric RNA (telRNA/TERRA) sequences compared to their DNA analogues, as studied by electrospray mass spectrometry ... [more ▼]

We report on the self-assembled structures formed by 12-mer, 22-mer, and 45-mer telomeric RNA (telRNA/TERRA) sequences compared to their DNA analogues, as studied by electrospray mass spectrometry, circular dichroism, and thermal denaturation. The major difference between telomeric RNA and DNA sequences is the ability of telomeric RNA to form higher-order dimeric assemblies, initiated by cation-mediated stacking of two parallel G-quadruplex subunits. The 5′-5′ stacking had been observed recently by NMR for the r(GGGUUAGGGU) 10-mer (Martadinata, H.; Phan, A. T. J. Am. Chem. Soc. 2009, 131, 2570); the present work shows that stacking also occurs for the 22-mer containing four G-tracts and for the 45-mer containing eight G-tracts, suggesting a general structural feature of telomeric RNA. The importance of kinetic effects in multimer formation, unfolding, and structural rearrangements is also highlighted. [less ▲]

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See detailZwitterionic i-motif structures are preserved in DNA negatively charged ions produced by electrospray mass spectrometry
Rosu, Frédéric ULg; Gabelica, Valérie ULg; Joly, Laure ULg et al

in Physical Chemistry Chemical Physics [=PCCP] (2010), 12

DNA cytosine-rich strands can fold into an intercalated motif (i-motif) structure. The i-motif is formed by mutually intercalated duplexes containing proton-mediated C-H(+)-C (cytosine-proton-cytosine ... [more ▼]

DNA cytosine-rich strands can fold into an intercalated motif (i-motif) structure. The i-motif is formed by mutually intercalated duplexes containing proton-mediated C-H(+)-C (cytosine-proton-cytosine) base pairs. Negatively charged ions of DNA i-motifs produced by electrospray mass spectrometry are therefore zwitterionic if the base pairing motif is preserved in the gas phase. Here we used IRMPD spectroscopy and ion mobility spectrometry to assess whether i-motif structures were preserved in the gas phase. We first investigated the IRMPD spectral signature of the tetramer [dC(6)](4), which can only be formed via C-H(+)-C base pairing, compared to the single strand dC(6). The IR signature of i-motif formation is an apparent broadening of the band at 1650 cm(-1). DFT calculations show this apparent broadening is actually due to blue-shifts of the NH(2) scissoring modes and red shifts of C[double bond, length as m-dash]O stretching modes. We then investigated the gas-phase conformations of the telomeric sequence d(CCCAAT)(3)CCC, that can form an intramolecular i-motif, by performing IRMPD spectroscopy and ion mobility spectrometry as a function of the charge state. We show that the negative ions of the lowest charge states correspond to a preserved i-motif structure. This is the first demonstration of the native extraction of solution-phase zwitterionic nucleic acids using negative electrospray ionization. [less ▲]

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See detailA simple method for determination of electrospray response factors of noncovalent complexes: application to DNA G-quadruplex binding and self-assembly
Gabelica, Valérie ULg; Amato, Jussara; Oliviero, Giorgia et al

Conference (2009, June 04)

Introduction Electrospray mass spectrometry (ESI-MS) is now widely used to investigate non-covalent assemblies, and the power of the method resides in the individual detection of the various ... [more ▼]

Introduction Electrospray mass spectrometry (ESI-MS) is now widely used to investigate non-covalent assemblies, and the power of the method resides in the individual detection of the various stoichiometries present in the mixture. However, to characterize the mixture quantitatively (i.e. to determine the concentration of each constituent), the biggest challenge comes from the fact that electrospray response factors of all constituents can be different, especially for constituents of significantly different masses and charges. Here we present a novel and simple method to determine the response factors of all constituents, using an internal standard. The method can be transposed to a wide variety of biological problems and to different ionization methods. Experimental All oligodeoxynucleotides were purchased from Eurogentec (Seraing, Belgium). G-quadruplexes formation is induced by addition of ammonium acetate (150 mM) to the guanine-rich strand. Polythymines (e.g. dT6) with m/z not overlapping with those of the complexes were used as internal standards. The ESI mass spectra were acquired using a Q-TOF Ultima Global (Waters, Manchester, UK) in the negative ion mode, with source parameters optimized for detecting the intact complexes. Quadruplex-ligand binding constants were quantified by titration of the quadruplex with the ligand (BOQ1, Dr. Teulade-Fichou). The kinetics of G-quadruplex self-assembly was monitored on-line, with the starting time defined by the addition of ammonium to the single strand. Peak areas were calculated using MassLynx and data were processed using Mathcad. Preliminary results Let us illustrate the method for the characterization of the self-assembly of dCGGTGGT. In NH4OAc, this G-rich strand forms a tetramer within a few hours, but the most stable species is an octamer, which is formed after a few days. The relative intensities of monomer, tetramer and octamer vary with the experimental settings: harsher conditions favor the transmission of the larger complexes. It is therefore foolish to assume that the response factors of the monomer, tetramer and octamer are equal. Using dT6 as an internal reference, we monitored the self-assembly kinetics by recording at several time points the following intensities: I(ref2-), I(G12-), I(G45-) and I(G89-), where Gn indicates a complex containing n G-rich strands. For each species Gn, we define Rn as its response factor relative to that of the reference: [Gn]/[ref] = Rn * I(Gn)/I(ref). At each time point the mass balance equation for the strand concentration must be satisfied: [G]tot = [G1] + 4[G4] + 8[G8]. Substituting with the definition of Rn’s one obtains a set of equations: [G]tot = [ref] * {R1*(I(G1)/I(ref) + 4R4*I(G4)/I(ref) + 8R8*I(G8)/I(ref)}. If the number of equations (time points) is greater than the number of unknown response factors, the least-squares fit to this system of equations is calculated using the Moore-Penrose pseudoinverse of the matrix constituted by the intensity ratios. Once all response factors are known, concentrations are recalculated at each time point, providing detailed characterization of the reaction kinetics, including reaction intermediates. We will also show that the same method can be used to quantify ligand binding using titration of the substrate by the ligand. This will be illustrated with titration of the quadruplex (TGGGGT)4 by ligand BOQ1. This ligand binds to the quadruplex and also produces ligand-bridged quadruplex multimers, which the present method allows quantifying for the first time. [less ▲]

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See detailPutative DNA G-quadruplex formation within the promoters of Plasmodium falciparum var genes
Smargiasso, Nicolas ULg; Gabelica, Valérie ULg; Damblon, Christian ULg et al

in BMC Genomics (2009), 10

Background. Guanine-rich nucleic acid sequences are capable of folding into an intramolecular four-stranded structure called a G-quadruplex. When found in gene promoter regions, G-quadruplexes can ... [more ▼]

Background. Guanine-rich nucleic acid sequences are capable of folding into an intramolecular four-stranded structure called a G-quadruplex. When found in gene promoter regions, G-quadruplexes can downregulate gene expression, possibly by blocking the transcriptional machinery. Here we have used a genome-wide bioinformatic approach to identify Putative G-Quadruplex Sequences (PQS) in the Plasmodium falciparum genome, along with biophysical techniques to examine the physiological stability of P. falciparum PQS in vitro. Results. We identified 63 PQS in the non-telomeric regions of the P. falciparum clone 3D7. Interestingly, 16 of these PQS occurred in the upstream region of a subset of the P. falciparum var genes (group B var genes). The var gene family encodes PfEMP1, the parasite’s major variant antigen and adhesin expressed at the surface of infected erythrocytes, that plays a key role in malaria pathogenesis and immune evasion. The ability of the PQS found in the upstream regions of group B var genes (UpsB-Q) to form stable Gquadruplex structures in vitro was confirmed using 1H NMR, circular dichroism, UV spectroscopy, and thermal denaturation experiments. Moreover, the synthetic compound BOQ1 that shows a higher affinity for DNA forming quadruplex rather than duplex structures was found to bind with high affinity to the UpsB-Q. Conclusions. This is the first demonstration of non-telomeric PQS in the genome of P. falciparum that form stable G-quadruplexes under physiological conditions in vitro. These results allow the generation of a novel hypothesis that the G-quadruplex sequences in the upstream regions of var genes have the potential to play a role in the transcriptional control of this major virulence-associated multi-gene family. [less ▲]

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See detailA Simple Method to Determine Electrospray Response Factors of Noncovalent Complexes
Gabelica, Valérie ULg; Rosu, Frédéric ULg; De Pauw, Edwin ULg

in Analytical Chemistry (2009), 81(16), 6708-6715

The quantitative study of noncovalent complexes by electrospray mass spectrometry requires the determination of the relative response of each species. The method proposed here to determine the ... [more ▼]

The quantitative study of noncovalent complexes by electrospray mass spectrometry requires the determination of the relative response of each species. The method proposed here to determine the electrospray response factors is based on the use of (1) an internal standard and (2) the mass balance equation applied to one binding partner M, for which different complexes MxLy are detected in the electrospray mass spectra. A set of experiments providing various ratios between the complexes (e.g. different ligand concentrations in a titration experiment or different time points in a kinetics experiment) is used to generate a set of independent linear equations that can be solved using simple matrix algebra to find the response factors of each MxLy complex relative to that of the internal standard. The response factors can then be used to determine equilibrium dissociation constants or for the quantitative monitoring of reaction kinetics. The first is illustrated with a study of DNA-ligand complexes, where we show that neither minor groove binding nor intercalation dramatically affects the DNA response factor. The second is illustrated with a study of the association kinetics of the telomeric G-quadruplex dGGG(TTAGGG)3 with its complementary strand, where the response factors allow correcting for the relative response of the quadruplex and the long duplex and obtaining reproducible association rate constants independently of the source tuning potentials. [less ▲]

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See detailIR and UV spectroscopic signatures of DNA higher-order structures in the gas phase
Gabelica, Valérie ULg; Rosu, Frédéric ULg; Gregoire, Gilles et al

Conference (2008, June 03)

Introduction Electrospray mass spectrometry (ESI-MS) can be used to transfer large biomolecular complexes from the solution to the gas phase. However, a longstanding question is whether the gas-phase ... [more ▼]

Introduction Electrospray mass spectrometry (ESI-MS) can be used to transfer large biomolecular complexes from the solution to the gas phase. However, a longstanding question is whether the gas-phase multiply-charged ions produced by ESI-MS keep a folded conformation in the absence of solvent. Nucleic acid secondary structures are determined by hydrogen bonding interactions between nucleic bases and by stacking interactions between neighboring base pairs. Here we will show that infrared (IR) and ultraviolet (UV) action spectroscopies provide useful and complementary information on the structure of nucleic acid ions in the gas phase. Methods IR spectroscopy experiments on DNA negative ions were carried out at the CLIO free electron laser (FEL) center (Orsay, France) using an Esquire 3000 (Bruker) mass spectrometer modified to inject the IR beam through the ring electrode. IRMPD spectra are recorded by monitoring the fragmentation of mass-selected parent ions as a function of the excitation wavenumber, in the range 1000-2000 cm-1. UV spectroscopy experiments were carried out using a tunable OPO laser (Continuum Lasers) with frequency doubling. The laser is interfaced with either a Finnigan LCQ ESI-QIT mass spectrometer or a Bruker Apex-Qe 9.4 T ESI-FTICR mass spectrometer. The UV action spectra were recorded by monitoring electron detachment as a function of the wavelength between 220 and 300 nm. Preliminary results First, DNA oligonucleotide ions forming G-quadruplex structures were studied in the gas phase using IR multiple-photon dissociation spectroscopy. Data interpretation on these large biomolecule ions is made using carefully chosen control experiments. The IR spectrum of the (dTG4T)4 quadruplex has been recorded, and compared to that of the single strand. Given the strand stoichiometry and the selective incorporation of three ammonium cations, there is little doubt about the quadruplex structure of [(dTG4T)4•(NH4+)3]5-. The major finding is a fingerprint of hydrogen bonding in the gas phase in the guanine C6=O6 stretching mode, that allows probing the conservation of G-quartets in the gas phase. Further experiments also demonstrate the conservation of G-quadruplex hydrogen bonds in the human telomeric sequence d(TTAGGG)4 [Gabelica et al., JACS, accepted]. Second, we also studied DNA duplexes and G-quadruplex ions in the gas phase by UV spectroscopy. We recorded the UV spectra of the (dTG4T)4 quadruplex, with and without ammonium ions. Molecular modeling [Rueda et al., JACS, 2006, p3608] and ion mobility spectrometry data [Gabelica et al., JACS, 2007, 895] showed that G-quadruplexes keep their hydrogen-bonded structure but become more floppy if inner cations are removed. We found that the UV spectra differ dramatically with and without inner cations, suggesting that UV spectroscopy is very sensitive to stacking interactions between neighboring G-quartets. We also used UV spectroscopy to probe the structure of 12-mer DNA duplexes, by comparing the duplex spectra to those obtained on single strands. Preliminary results show that stacking interactions may be preserved in duplexes containing GC base pairs, but not in duplexes containing AT base pairs. Altogether, these results show the complementarities between IR and UV spectroscopy to characterize DNA structures in the gas phase: IR data mainly give access to information on hydrogen bonding of bases, and UV spectroscopy provides information on stacking interactions. [less ▲]

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See detailElectron Photodetachment of DNA Polyanions: Photoelectron Spectroscopy and UV Action Spectroscopy
Gabelica, Valérie ULg; Rosu, Frédéric ULg; De Pauw, Edwin ULg et al

Conference (2008, April 15)

DNA polyanions trapped in a mass spectrometer undergo electron detachment following irradiation with UV light [1-3]. Electron photodetachment is a 1-photon process, and its efficiency depends on: - The ... [more ▼]

DNA polyanions trapped in a mass spectrometer undergo electron detachment following irradiation with UV light [1-3]. Electron photodetachment is a 1-photon process, and its efficiency depends on: - The nature of the DNA bases: guanine-containing strands are the most prone to electron photodetachment, followed by adenine, cytosine, and finally thymine. - The excitation wavelength: electron detachment is maximum around 260 nm, corresponding to base excitation. - The charge of the polyanion: higher charge state ions undergo more efficient electron detachment because of the Coulombic repulsion. Here we will discuss the electron photodetachment mechanism in the light of the most recent experimental results. Because the base-dependence of electron photodetachment efficiency is correlated with the base ionization potential and is maximum at wavelengths corresponding to the base absorption, we initially proposed that electron photodetachment might occur directly from the base, and that the photodetachment yield was correlated with the electron binding energy to the base [2]. Photoelectron spectroscopy experiments were performed on DNA multiply charged anions with varying base composition to probe how the electron binding energies changes with the base composition. Finally, the electron detachment channel was used to perform UV spectroscopy experiments on large DNA polyanions trapped in the gas phase. Gas-phase UV spectra of DNA duplexes and G-quadruplexes containing up to 24 bases (> 7000 Da) will be presented. [1] V. Gabelica, T. Tabarin, R. Antoine, F. Rosu, I. Compagnon, M. Broyer, E. De Pauw, and P. Dugourd, Anal. Chem. 78, 6564 (2006). [2] V. Gabelica, F. Rosu, T. Tabarin, C. Kinet, R. Antoine, M. Broyer, E. De Pauw, and P. Dugourd, J. Am. Chem. Soc. 129, 4706 (2007). [3] V. Gabelica, F. Rosu, E. De Pauw, R. Antoine, T. Tabarin, M. Broyer, and P. Dugourd, J. Am. Soc. Mass Spectrom. 18, 1990 (2007). [less ▲]

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See detailIR and UV spectroscopy of DNA ions stored in a quadrupole ion trap mass spectrometer
Gabelica, Valérie ULg; Rosu, Frédéric ULg; Gregoire, Gilles et al

Conference (2008, February 15)

Electrospray mass spectrometry (ESI-MS) can be used to transfer large biomolecular complexes from the solution to the gas phase. However, a longstanding question is whether the gas-phase multiply-charged ... [more ▼]

Electrospray mass spectrometry (ESI-MS) can be used to transfer large biomolecular complexes from the solution to the gas phase. However, a longstanding question is whether the gas-phase multiply-charged ions produced by ESI-MS keep a folded conformation in the absence of solvent. Nucleic acid secondary structures are determined by hydrogen bonding interactions between nucleic bases and by stacking interactions between neighboring base pairs. In solution, infrared (IR) and ultraviolet (UV) spectroscopies provide information on hydrogen bonding and stacking interactions in nucleic acids, respectively. Here we will show how IR and UV spectra of gas-phase ions can be recorded, and what can be learned on the structure of nucleic acids (double helices and quadruple helices) in the gas phase. The IR spectroscopy experiments on DNA negative ions were carried out at the CLIO free electron laser (FEL) center using an electrospray quadrupole ion trap mass spectrometer (Esquire 3000, Bruker Daltonics, Germany) modified to inject the IR beam in the trap through the ring electrode. IRMPD spectra are recorded by monitoring the relative fragmentation efficiency of mass-selected parent ions as a function of the excitation wavenumber, in the range 1000-2000 cm-1. Data interpretation on these large biomolecule ions is made using carefully chosen control experiments. The major finding is a fingerprint of hydrogen bonding in the gas phase in the guanine C6=O6 stretching mode, that allows probing the conservation of G-quartets in the gas phase. The experiments demonstrate the conservation of G-quadruplex hydrogen bonds in the human telomeric sequence d(TTAGGG)4. The UV spectroscopy experiments were carried out using a tunable OPO laser (Continuum Lasers, Santa Clara, CA, USA) with frequency doubling. The laser is interfaced with a Finnigan LCQ ESI-QIT mass spectrometer. The UV action spectra were recorded by monitoring electron detachment from DNA multiply charged anions as a function of the wavelength between 220 and 300 nm. Preliminary results suggest that stacking interactions are preserved in duplexes containing GC base pairs, and in G-quadruplexes containing inner cations. [less ▲]

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See detailProteome alteration induced by hTERT transfection of human fibroblast cells
Mazzucchelli, Gabriel ULg; Gabelica, Valérie ULg; Smargiasso, Nicolas ULg et al

in Proteome Science (2008), 6(1), 12

Background: Telomerase confers cellular immortality by elongating telomeres, thereby circumventing the Hayflick limit. Extended-life-span cells have been generated by transfection with the human ... [more ▼]

Background: Telomerase confers cellular immortality by elongating telomeres, thereby circumventing the Hayflick limit. Extended-life-span cells have been generated by transfection with the human telomerase reverse transcriptase (hTERT) gene. hTERT transfected cell lines may be of outstanding interest to monitor the effect of drugs targeting the telomerase activity. The incidence of hTERT gene transfection at the proteome level is a prerequisite to that purpose. The effect of the transfection has been studied on the proteome of human fibroblast (W138). Cytosolic and nuclear fractions of W138 cells, empty vector transfected W138 (W138-HPV) and hTERT W138 cells were submitted to a 2D-DIGE (Two-Dimensional Differential In-Gel Electrophoresis) analysis. Only spots that had a similar abundance in W138 and W138-HPV, but were differentially expressed in W138 hTERT were selected for MS identification. This method directly points to the proteins linked with the hTERT expression. Number of false positive differentially expressed proteins has been excluded by using control W138-HPV cells. The proteome alteration induced by hTERT W138 transfection should be taken into account in subsequent use of the cell line for anti-telomerase drugs evaluation. Results: 2D-DIGE experiment shows that 57 spots out of 2246 are significantly differentially expressed in the cytosolic fraction due to hTERT transfection, and 38 were confidently identified. In the nuclear fraction, 44 spots out of 2172 were selected in the differential proteome analysis, and 14 were identified. The results show that, in addition to elongating telomeres, hTERT gene transfection has other physiological roles, among which an enhanced ER capacity and a potent cell protection against apoptosis. Conclusion: We show that the methodology reduces the complexity of the proteome analysis and highlights proteins implicated in other processes than telomere elongation. hTERT induced proteome changes suggest that telomerase expression enhances natural cell repair mechanisms and stress resistance probably required for long term resistance of immortalized cells. Thus, hTERT transfected cells can not be only consider as an immortal equivalent to parental cells but also as cells which are over-resistant to stresses. These findings are the prerequisite for any larger proteomics aiming to evaluate anti-telomerase drugs proteome alteration and thus therapeutics induced cell reactions. [less ▲]

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